A unifying mechanism for the biogenesis of prokaryotic membrane proteins co-operatively integrated by the Sec and Tat pathways

The vast majority of polytopic membrane proteins are inserted into the cytoplasmic membrane of prokaryotes by the general secretory (Sec) pathway. However, a subset of monotopic proteins that contain non-covalently-bound redox cofactors depend on the twin-arginine translocase (Tat) machinery for membrane integration. Recently actinobacterial Rieske iron-sulfur cluster-containing proteins were identified as an unusual class of membrane proteins that require both the Sec and Tat pathways for the insertion of their three transmembrane domains (TMDs). The Sec pathway inserts the first two TMDs of these proteins co-translationally, but releases the polypeptide prior to the integration of TMD3 to allow folding of the cofactor-containing domain and its translocation by Tat. Here we have investigated features of the Streptomyces coelicolor Rieske polypeptide that modulate its interaction with the Sec and Tat machineries. Mutagenesis of a highly conserved loop region between Sec-dependent TMD2 and Tat-dependent TMD3 shows that it plays no significant role in coordinating the activities of the two translocases, but that a minimum loop length of approximately eight amino acids is required for the Tat machinery to recognise TMD3. Instead we show that a combination of relatively low hydrophobicity of TMD3, coupled with the presence of C-terminal positively-charged amino acids, results in abortive insertion of TMD3 by the Sec pathway and its release at the cytoplasmic side of the membrane. Bioinformatic analysis identified two further families of polytopic membrane proteins that share features of dual Sec-Tat-targeted membrane proteins. A predicted heme-molybdenum cofactor-containing protein with five TMDs, and a polyferredoxin also with five predicted TMDs, are encoded across bacterial and archaeal genomes. We demonstrate that membrane insertion of representatives of each of these newly-identified protein families is dependent on more than one protein translocase, with the Tat machinery recognising TMD5. Importantly, the combination of low hydrophobicity of the final TMD and the presence of multiple C-terminal positive charges that serve as critical Sec-release features for the actinobacterial Rieske protein also dictate Sec release in these further protein families. Therefore we conclude that a simple unifying mechanism governs the assembly of dual targeted membrane proteins.


Introduction
Prokaryotic cytoplasmic membrane proteins represent 20-30% of the proteome (1, 2) and they fulfil a wide variety of critical functions in the cell including respiration, photosynthesis, and ion transport, allowing this membrane to act as a tightly controlled barrier between the cytoplasm and the extracellular environment. Cytoplasmic integral membrane proteins adopt α-helical topologies, and in bacteria are inserted via the action of at least one of three protein translocation machineries -the Sec machinery, the YidC insertase and the Tat pathway (see (3) for a recent review).
The SecYEG translocon is the major route by which multi-spanning membrane proteins are integrated into the membrane. The insertion of transmembrane domains of polytopic proteins occurs co-translationally following targeting of the translating ribosome to the Sec machinery through the action of signal recognition particle (SRP) (4). YidC is positioned close to the lateral gate of SecY and interacts with nascent transmembrane domains to facilitate their integration into the membrane (5-7). YidC can also act independently of the Sec system to integrate small (usually mono-or bitopic) membrane proteins directly into the bilayer (8,9). The final topology adopted by a polytopic membrane protein depends upon a number of intrinsic and extrinsic factors including the hydrophobicity of membrane-spanning regions, the number and location of positively-charged amino acids and the composition of the lipid bilayer (10-12).
The Tat system is a post-translational protein transport pathway that operates independently of the Sec and YidC machineries to transport folded proteins across the cytoplasmic membrane (reviewed in 13, 14). Proteins are targeted to the Tat machinery by N-terminal signal sequences containing a highly conserved pair of arginine residues that are usually critical for efficient recognition of substrates (15). A subset of Tat substrate proteins contain non-covalently bound prosthetic groups such as metal-sulphur clusters or nucleotide-based cofactors, many of which play important roles in respiratory and photosynthetic metabolism (16). Some Tat substrates are also integral membrane proteins. In bacteria Tat-dependent integral membrane proteins generally fall into two classes -those that are N-terminally anchored in the bilayer by a non-cleaved signal sequence, such as the Rieske iron-sulfur proteins for example of Paracoccus or Legionella (17,18) or the TtrA subunit of Salmonella tetrathionate reductase (19) and those that have a single transmembrane helix at their Ctermini such as the small subunits of hydrogenases and formate dehydrogenases (20,21).
Recent studies have indicated that the Rieske proteins of actinobacteria are highly unusual Tat substrates (22,23). Rieske proteins are essential membrane-bound components of cytochrome bc 1 and b 6 f complexes that coordinate an iron-sulfur (FeS) cluster involved in the electron transfer from quinones to cytochromes c 1 /f (for reviews see (24,25)). The actinobacterial proteins have three transmembrane domains (TMDs) preceding the Rieske FeS domain, unlike most other Rieske proteins which contain only one TMD. Inspection of actinobacterial Rieske sequences indicates the presence of a predicted twin-arginine motif between TMDs 2 and 3, suggesting the possibility that the concerted action of more than one translocase may be required for correct assembly. Indeed it was shown that the first two TMDs of the Streptomyces coelicolor Rieske protein, Sco2149, are inserted by the Sec machinery, probably in a co-translational manner, whereas the insertion of TMD3 is dependent on the Tat pathway (22), providing the first example of these two machineries operating together to assemble a single protein.
These findings raise a number of pertinent questions about the mechanisms by which these translocases are co-ordinated to ensure that the Sec system does not integrate TMD3 but releases the polypeptide to allow folding of the globular domain, and the subsequent recognition of a membrane-tethered substrate by the Tat pathway. It also raises the question whether actinobacterial Rieske proteins represent an oddity of nature, or whether there are further examples of dual Sec/Tat-targeted membrane proteins to be discovered. Here we have addressed both of these major aspects and show that in addition to Rieske there are at least two further conserved families of dual targeted membrane proteins across bacteria and archaea that each have 5 TMDs. A detailed dissection of the features of the transmembrane regions of S. coelicolor Rieske reveals that the relatively low hydrophobicity of TMD3 coupled with the location of positively charged amino acid residues orchestrate the release of the polypeptide by the Sec pathway. Importantly, we demonstrate that these features are also present across all identified families of these dual-targeted membrane proteins indicating that there is unifying mechanism for their biogenesis.

Fusion proteins for the analysis of Sco2149 membrane assembly.
Previous work has shown that the S. coelicolor Rieske protein, Sco2149, has three transmembrane domains that require the combined action of two distinct protein translocases, Sec and Tat, for complete assembly into the membrane (22,23). However the mechanism by which these two translocases are coordinated is unknown, although TMD and globular domain swapping experiments indicated that the information required to coordinate this process does not reside within the first two TMDs or the cofactor binding domain (22).
To assess the mechanism of TMD insertion we used constructs where the cofactor-containing FeS domain was genetically removed from Sco2149 and replaced with the mature region of two different reporter proteins -that of the E. coli Tat substrate AmiA (26) to report on interaction of Sco2149 with the Tat pathway, or of the Sec substrate -lactamase (Bla, which is compatible for export with either the Sec or Tat pathways depending on the nature of the targeting sequence (27)) ( Fig 1A, Fig S1). These constructs were produced from the medium copy number vector pSU-PROM (which specifies kanamycin resistance (28)) under control of the constitutive tatA promoter (29).
AmiA and its homologue AmiC are periplasmic Tat substrates that remodel the peptidoglycan, and in their absence E. coli is sensitive to growth in the presence of SDS (26, 30) ( Fig 1C; top panel). As expected, when either plasmid-encoded native AmiA or the Sco2149 TMD -AmiA fusion was produced in the tat + strain lacking chromosomally encoded periplasmic AmiA and AmiC (MCDSSAC), growth on SDS was restored ( Fig 1C, middle two panels). The export of AmiA from both of these constructs was absolutely dependent on the Tat pathway as no growth on SDS was conferred in the tatstrain (MCDSSAC tat). Previously it has been reported that a twin lysine substitution of the twin arginine motif of Sco2149 was sufficient to prevent Tat-dependent export of AmiA when produced at lower levels from the pSU18 plasmid (22). However, when expressed from the pSU-PROM vector, a low level of export by the Tat pathway could still be observed for the Sco2149-AmiA construct harbouring this substitution ( Fig S2). It has been noted previously that Tat-dependent export of some very sensitive plasmid-borne reporter proteins can be detected following twin lysine substitution of the twin arginines (31, 32), indicating that twin lysines can still trigger Tat-dependent export but with a greatly reduced efficiency. However, less conservative substitutions of the twin arginine motif to twin alanine or to alanine-aspartate were not permissive for Tat transport (Fig 1C; Fig S2).
The membrane insertion of Sco2149 was further investigated using the Bla fusion construct. When exported to the periplasmic side of the membrane Bla confers resistance to ampicillin, which can be assessed in a quantitative manner using M.I.C.Evaluator TM test strips. Fig 1D shows that the basal M.I.C. for ampicillin was evaluated at 2.5 and 1.4 µg/ml, respectively, for the tat + (MC4100) and tat -(DADE) strains harbouring the empty vector. We assign these slight differences in M.I.C. to the partially compromised cell wall in tat mutant strains (26,30). The tat + strain producing the Sco2149 TMD -Bla fusion protein was able to grow up to a concentration of approximately 15 µg/ml ampicillin, indicating that there was export of Bla in this strain. However, some of that export was clearly by the Sec pathway since the tatstrain producing Sco2149 TMD -Bla had an M.I.C. for ampicillin of 7.6 µg/ml, significantly above basal level. It has been reported that the introduction of negative charges into the n-region of a Sec signal peptide blocks Sec-dependent translocation (33), and therefore substituting the twin arginines to alanine-aspartate would be expected to prevent translocation through both the Sec and Tat pathways. As shown in Fig 1D these substitutions reduced the MIC for ampicillin to 4.0 and 1.3 µg/ml, respectively, for tat + and tatstrain, very close to basal level.
Taken together these results indicate that there is some compatibility of TMD3 of the S.
coelicolor Rieske protein with the Sec pathway, which was not seen previously using a more qualitative assay (22).

The cytoplasmic loop region of Sco2149 does not modulate interaction of TMD3 with
the Sec pathway.
The finding that there is some Sec-dependent translocation of the Bla portion of the Sco2149 TMD -Bla fusion in a strain lacking the Tat pathway provides a useful tool to study features of the protein that influence interaction with the Sec machinery. We therefore undertook a programme of mutagenesis on the Sco2149 TMD -Bla construct, focusing firstly on the cytoplasmic loop region between TMD2 and TMD3 as this has a number of highly conserved features across actinobacterial Rieske proteins (Fig 1B; Fig S3). In particular the loop has a highly conserved length (43 amino acids between the predicted end of TM2 and the twin arginine motif), a region of predicted -helical structure, and a number of positions where positively or negatively charged residues are conserved, including an almost invariant glutamic acid (E127 in Sco2149) and arginine-histidine pairing (R133, H134 in Sco2149).
Initial site-directed replacement of amino acids in the loop region were undertaken and the level of resistance to ampicillin mediated by the variant Sco2149 TMD -Bla fusion protein in a tatbackground was scored. As shown in Table 1, apart from the introduction of an alanineaspartate pair to replace the twin arginines, none of the substitutions we introduced, including replacement of the highly conserved E127 or R133/H134 residues or introduction of proline residues into the predicted -helical region, had any substantive effect on the interaction of Sco2149 with the Sec pathway. We therefore made further substitutions, for example progressively deleting clusters of negatively charged amino acids or changing them to positively charged lysines. None of these deletions or substitutions had any detectable effect on Sec translocation of the Bla fusion, even when all of the acidic residues were substituted for lysine. Moreover, insertion of three additional negative charges into the loop was also without detectable effect.
We similarly assessed translocation by Sec for a series of sliding truncations of 5, 10, 15, 20, 25, 30 and 35 residues within the loop region (summarised in Table 2). Again most of the truncations had little effect on translocation of Sco2149 TMD -Bla by the Sec pathway, and even truncations of 30 residues or more gave mean M.I.C.s for ampicillin similar to that seen for the non-mutated construct. These findings indicate that many of the conserved features noted in this loop region, for example the overall length, presence of a predicted -helical region and clusters of negatively charged amino acids do not modulate interaction of Sco2149 with the Sec pathway.
We did note, however, that one of the 35 residue truncations, ∆123-157, significantly reduced integration of TMD3 by the Sec pathway (Fig 2A,B), whereas the other 35 residue truncation, ∆118-152, showed a slight increase in Sec translocation (c.f. M.I.C of 7.6 g/ml ampicillin for the non-mutated construct vs 12g/ml for the ∆118-152 truncation). This suggested that there may be some feature of the loop region between residues 153 and 157 influencing interaction with the Sec pathway. To explore this further we made a series of additional one amino acid truncations to give ∆118-153, ∆118-154, ∆118-155 and ∆118-156 and ∆118-157 constructs. A minimum cytoplasmic loop length is necessary for Tat recognition of Sco2149 TMD3.
Since none of the conserved features in the Sco2149 cytoplasmic loop were required for modulating interaction with the Sec pathway, we next addressed whether they were required for recognition by the Tat system. A subset of the amino acid substitutions and each of the sliding truncations was introduced into the Sco2149 TMD -AmiA fusion protein and expressed in a tat + strain to allow Tat-dependence to be scored by testing for growth in the presence of SDS (Tables 1, 2). Table 1 shows that, apart from substitutions at the twin arginine motif, none of the other variants affected Tat-dependent export of AmiA, including the introduction of prolines within the predicted -helical structure, or substitution of the highly conserved E127 or R133/H134. These results suggest that none of these features are required for recognition of the loop region by the Tat pathway.
Ordinarily, Tat signal peptides have free N-termini, whereas the Tat signal sequence of Sco2149 is internal and is only recognised by the Tat pathway once the first 2 TMD of the protein have been integrated by Sec. The loop truncation experiments indicated that the Tat system was still able to identify and integrate TMD3 when it was truncated by up to 30 residues. However, one of the 35 residue truncations (Sco2149 TMD -∆123-157-AmiA) and the 40 residue truncation (Sco2149 TMD -∆118-157-AmiA) supported no growth on SDS-containing media (Table 2), indicating that there is a minimum loop length requirement of approximately eight amino acids between TMD2 and the twin arginine motif is required for Tat recognition of a tethered signal peptide.
Taken together we conclude that, with the exception of the twin arginine motif, none of the conserved features of cytoplasmic loop are strictly necessary for interaction of Sco2149 with the Tat pathway or to mediate release from Sec.

Specific physical properties of TMD3 drive its release from Sec.
Hydrophobicity is the driving force for the insertion of a helix into the membrane (10, 36, 37).
Analysis of transmembrane helices from polytopic proteins of known three-dimensional structure shows a general trend that the first and last TMDs are of similar hydrophobicity, and they are notably more hydrophobic than the central helices (38,39). An analysis of the apparent G for the insertion of the three TMDs of selected actinobacterial Rieske proteins is shown in Table 3. It can be seen that the first and second TMDs have negative predicted G app values and are therefore expected to be inserted as TMDs by the Sec system (40). However, the third and final TMD is predicted to have a positive G app (Table 3). This is in contrast to the final TMD of 'standard' Sec-dependent proteins and suggests that this helix might be poorly recognised by the Sec machinery.
To probe this further we investigated the effect of increasing the hydrophobicity of TMD3. Table 3 shows that substitution of a single leucine residue at either serine 179 or glycine 180 reduces the predicted G app value for TMD3 Sec-dependent membrane insertion by at least 0.6 kcal mol -1 Accordingly, when these single substitutions were individually introduced into the Sco2149 TMD -Bla fusion in tatcells, a dramatic increase in M.I.C for ampicillin of up to 25 fold was observed (Fig 3B), almost at the upper limit of detection. Combining these substitutions (S179L, G180L), and including a third substitution (P177L) shifts the predicted G app value closer to that of TMD1 (Table 1). These substitutions also significantly increased the observed M.I.C. over the unsubstituted fusion, but did not appear to have additive effects over the single leucine substitutions. We conclude that the low hydrophobicity of TMD3 is a key driver for the release of Sco2149 from the Sec machinery.
It has long been known that Tat signal peptides frequently contain one or more positive charges in their c-regions, close to the site of signal peptidase cleavage. These charges are not required for the interaction with the Tat pathway but reduce the efficiency of interaction with Sec and have therefore been described ' . A positive charge is generally also found close to the C-terminal end of TMD3 of actinobacterial Rieske proteins (R185 in the case of Sco2149; Fig 3A, Fig S1). Substitution of R185 for alanine in the Sco2149 TMD -Bla fusion conferred an 8-fold increase in M.I.C for ampicillin, and therefore R185 also appears to act as a Sec-avoidance motif in this context. Interestingly, closer inspection of actinobacterial Rieske proteins indicates that there are a number of further non-conserved positive charges located within the C-terminal vicinity of TMD3 (Fig 3A underlined residues, Fig S1 orange residues) which are not found in other Rieske proteins that only contain a single TMD ( Fig S1B). Since our original Sco2149 TMD -Bla fusion (where the Bla sequence is fused immediately after R185) lacks most of these additional charges (Fig 3A), we made an additional Bla fusion where the Sco2149 sequence in the fusion protein was extended to aa205, incorporating an additional four positively charged residues. It can be seen that inclusion of this additional positively charged stretch almost completely abolished transport via Sec, as the clearance zone around the M.I.C. strip was of similar size to that of the negative control ( Fig 3C). We did, however, note that for unknown reasons there was a variable level of breakthrough growth within the zone of clearing for strain DADE producing the extended Sco2149 TMD -Bla fusion. We therefore constructed similar Bla fusions after TMD3 of the M.
tuberculosis Rieske protein, QcrA. Fig 3D indicates that there is some Sec-dependent export of the Bla fusion when it is fused close to the C-terminal end ('short fusion') but that this was almost abolished when the sequence was extended to introduce the positively charged stretch ('long fusion'). Taken together, we conclude that a combination of low hydrophobicity of TMD3 coupled with the presence of several C-terminal positive charges promotes release of actinobacterial Rieske proteins from the Sec machinery.

Bioinformatic analysis identifies further families of membrane proteins potentially dependent on both Sec and Tat pathways.
We next asked whether actinobacterial Rieske proteins were the only protein family that required both Sec and Tat pathways for their integration. To this end, all proteins from prokaryotic genomes available in Genbank were analysed by both TATFind 1.4 (44) and TMHMM 2.0c (2) programs. For each protein, both outputs were combined to identify the position of twin arginine motif, and the number of transmembrane helices present N-terminal and C-terminal to it. The final output from this search was sorted to give those proteins that had a predicted even number of TMDs prior to the twin-arginine motif and that had a predicted single TMD immediately following the twin-arginine motif (available as supplementary online file at: http://www.lifesci.dundee.ac.uk/groups/tracy_palmer/docs/CombinedTATFindTMHMMoutput .docx). We subsequently manually searched this list to identify any proteins with a predicted C-terminal cofactor-binding domain.
From the output we identified a further actinobacterial Rieske homolog from Kitasatospora setae (KSE_30950) that is predicted to have five TMDs, with the twin arginine motif adjacent to TMD5. We also identified two further families of predicted metalloproteins that shared features of dual-inserted proteins (shown schematically in Fig 4A). Sco3746, also from S.
coelicolor is predicted to have five TMDs, with a predicted molybdenum cofactor (MoCo) binding domain at the C-terminus and conserved histidine residues in TMDs 2, 3 and 4 that are predicted to co-ordinate two heme b moieties ( Fig 4A). The twin arginine motif, which is conserved across homologous proteins ( Fig S4) directly precedes TMD5. Homologues of Sco3746 were identified across the actinobacteria, as well as in firmicutes, chloroflexi and euryarchaeota, and each carries a twin arginine motif directly preceding TMD5 (Examples from each phyla are shown in Fig S4). Protein Q1NSB0 from the delta proteobacterium MLMS-1 is also predicted to have five TMDs and to contain seven 4Fe-4S clusters, three at the cytoplasmic side and four at the extracellular side of the membrane ( Fig 4A; Fig S5). Again the conserved twin arginine motif directly precedes TMD5 and homologues of this protein are encoded in many prokaryotic genomes including those from the chloroflexi, nitrospirae and euryarchaeota phyla ( Fig S5).
Reporter proteins fused to Sco3746 or predicted polyferredoxin from MLMS-1 are translocated by the Tat pathway.
To confirm that these newly identified proteins were indeed Tat substrates, we designed To confirm that this translocation was dependent on the Tat pathway, the twinarginines of the Tat recognition motif were substituted for two lysines. This conservative substitution abolished maltose fermentation (Fig 4C), indicating that MBP translocation was dependent on the Tat pathway. Similar findings were made using the AmiA reporter fusions.
Fig 4D shows that, as expected, when either plasmid-encoded Sco3746 TMD -AmiA or PFD TMD -AmiA was produced in the tat + strain lacking native AmiA/C, growth on SDS was supported.
Export was dependent on the Tat pathway since growth on SDS was not supported in the tatstrain, or in the tat + strain if the twin arginine motif was substituted for twin lysine. We conclude that Sco3746 and PFD are dependent on the Tat pathway for their assembly.
Sco3746 TMD and PFD TMD fusions are stably inserted in the membrane in the absence of a functional Tat system.
We next determined whether these fusion proteins were stably inserted into the membrane. thus two additional non-specific bands were also detected by the MBP antibody for these samples. Substitution of the Tat consensus arginine pair for di-lysine did not detectably affect the amount of fusion proteins produced, nor their membrane localization, indicating that membrane insertion of each of these fusions occurred independently of the Tat system. This was confirmed by repeating the analysis in a tatstrain, where as expected the fusions were again detected exclusively in the membranes. Washing the membranes with 4M urea did not extract either protein (Fig 5B), indicating that they were integrally inserted into the membrane in the absence of the Tat pathway. This indicates the participation of a second protein translocase, almost certainly the Sec pathway, in the insertion of these proteins into the membrane.

Sco3746 TMD -MBP has five TMDs.
To confirm the predicted topology of the hydrophobic domain of Sco3746, we undertook a cysteine accessibility study. The Sco3746 TMD -MBP fusion is naturally devoid of cysteine residues. Guided by topology prediction programs we made three Cys substitutions (G14C, A137C and A219C) that are predicted to reside at the cytoplasmic side of the membrane and two (G84C and G171C) that are located in predicted extracellular loops (Fig 5C). We produced these constructs in a tat + strain and probed cysteine accessibility using the reagent methoxypolyethyleneglycol maleimide (MAL-PEG). This reagent, which has a mass of around 5000 Da, can pass through the outer membrane in the presence of EDTA, but is impermeable to the inner membrane. Fig 5D shows that the G84C and G171C variants of Sco3746 TMD -MBP clearly labelled with MAL-PEG in whole cells confirming that they are extracellular. By contrast, G14C, A137C and A219C variants were not labelled in whole cells but were labelled upon cell lysis, consistent with them having a cytoplasmic location. Taken together we conclude that the Sco3746 TMD portion of the Sco3746 TMD -MBP fusion has 5 TMDs.
A conserved mechanism regulates Sec-Tat transfer for three dual-targeted protein families.
Our prior results analysing the interaction of actinobacterial Rieske proteins with the Sec pathway indicated that a combination of low hydrophobicity of the Tat-dependent TMD coupled with the presence of positive charges close to the C-terminal end of that TMD promoted release of the polypeptide from the Sec pathway. We therefore inspected the sequences of Sco3746 homologues and of PFD proteins to see whether these features are conserved across protein families. Fig S4 shows Table 2).
We constructed 'short' (after aa 252) and 'long' (after aa 272) variants of Sco3746 TMD fused to Bla (Fig 6A), and expressed these in a tatstrain to score for Sec-translocation of TMD5. Fig   6B shows that for the short fusion there is some degree of insertion of TMD5 by the Sec pathway because the M.I.C. for ampicillin mediated by this construct is significantly higher than the basal level. Substitution of hydrophobic leucines into TMD5 is predicted to shift the G app for membrane insertion of TMD5 from positive to negative (Table 4) G app values for membrane insertion of the 5 TMDs was complicated by the observation that the iron-sulfur cluster binding regions were variably called as TMDs by some prediction programs. We therefore analysed only the first and fifth (Tat-dependent) TMDs (Table 5), and again it can be seen that the Tat-dependent TMD has a positive predicted G app .
To probe PFD interaction with the Sec pathway we designed 'short' (fused after aa 371) and 'long' (fused after aa 374) fusions of PFD TMD from delta proteobacterium MLMS-1 to Bla ( Fig   7A) and produced these in a tatstrain to score for Sec-translocation of TMD5. However, neither of these constructs mediated detectable export of -lactamase as the M.I.C. for ampicillin was almost indistinguishable from the negative control (Fig 7B,C). We attribute this to the relatively poor expression of the PFD fusion proteins (e.g. Fig 5A). Next we substituted two, or three, leucine residues into TMD5 of PFD in each of the fusions, which is predicted to lower the G app value for TMD5 membrane insertion (Table 5). In agreement with this, Fig 7B shows that these substitutions significantly increased the level of interaction of the short fusion with the Sec pathway, giving mean M.I.C.s for ampicillin of 9.3g/ml for the G354L, R358L and 12.0g/ml for the G354L, P355L, R358L substitutions, respectively. These same substitutions also increased the interaction of the long fusion with Sec as they also conferred some resistance to ampicillin, each giving a mean M.I.C. of 6.7 g/ml (Fig 7C). However it is clear that the same leucine substitutions confer lower levels of resistance to ampicillin when they are present in the long construct than when they are in the short construct (compare Fig   7B with Fig 7C). Since the shorter construct harbours one less positive charge at the Cterminal end of TMD5 we conclude that the additional positive charge present in the extended fusion reduces the level of membrane insertion by Sec. Taken together our results demonstrate that the mechanism of Sec release of the final TMD is conserved across all known families of dual Sec-Tat targeted membrane proteins.

Discussion
In a previous study we identified the actinobacterial Rieske FeS protein as the first protein known to be targeted to the plasma membrane by the dual action of the Sec and Tat translocases. The mechanism by which translocation is coordinated between the two pathways was not known, although a length-and sequence-conserved loop region between Sec-dependent TMD2 and Tat-dependent TMD3 was implicated in this process (22) (47). This raises the possibility that rather than being an exception, Sec interaction with Tat signal peptides is much more frequent, and that following abortive attempts at Sec-translocation, membrane-associated twin-arginine signal peptides are common substrates of the Tat pathway. In this context it should be noted that both thylakoid and E. coli Tat substrates interact with the membrane before subsequent interaction with Tat machinery (48-51).
Our work has shown that dual targeted Sec-Tat dependent membrane proteins are dispersed across two domains including Gram-negative and Gram-positive bacteria and euryarchaea, indicating that the biogenesis of dual-targeted membrane proteins is a common feature of prokaryotes. It is interesting to note that distant homologs of both the predicted heme-Moco relationship of such proteins and their corresponding genes raises the possibility that dualtargeted proteins arose during the course of evolution from separate polypeptides but adjacent genes. Alternatively, the ancestral proteins may have been single, dual-targeted polypeptides that subsequently separated in some organisms.
The amino acid sequences of all of the fusion proteins used in this study can be found in Supplementary Information. All plasmids used and generated in this study are listed in Table   S2 and all oligonucleotides are listed in  (22)) was cloned in as an XbaI-HindIII fragment.
The entire PFD TMD -MBP coding region was subsequently excised as an EcoRI-HindIII fragment and cloned into similarly digested pSU18 (59) to give pSU18 PFD TMD -MBP. To construct pSU18 PFD TMD -AmiA, the MBP coding region was excised and replaced with the AmiA coding region (as an XbaI/HindIII fragment from pSU-PROM Sco2149 TMD -AmiA). To construct the PFD TMD -Bla fusion (which covers up to aa371 of PFD), oligonucleotides SU18.1 and PFD TMD BlaRev were used to amplify the PFD coding sequence (with pSU18 PFD TMD -Bla as template). The product was digested with EcoRI and KpnI and ligated into similarly digested pSU18PROM Sco3746 TMD -Bla to generate PFD TMD -Bla. The PFD TMD coding sequence in this construct was further extended to residue 374 using oligonucleotides SU18.1 and PFD TMD extension and pSU18 PFD TMD -Bla as template. The resultant product was digested with EcoRI-KpnI and ligated into similarly digested pSU18 PFD TMD -Bla to generate pSU18 PFD TMD extended-Bla.
Site-directed mutagenesis was performed using the QuickChange TM method (Stratagene) according to manufacturer's instructions. Deletion mutants were generated from a modified QuickChange TM method adapted from (62). Briefly, forward and reverse primers were designed to remove up to 5 residues at a time, overlapping by 12 nucleotides upstream and downstream of the region to be deleted with an overhang of 12 nucleotides at either end. For truncations larger than 5 residues the template used, already contained a downstream deletion of all residues but the additional 5 residues to be removed. All constructs were verified by DNA sequencing.
Unless otherwise stated, E. coli strains were grown aerobically overnight at 37°C in Luria-

Bertani (LB) broth supplemented with appropriate antibiotic/s at the indicated final
concentrations -ampicillin (125 µg/ml), kanamycin (50 µg/ml), apramycin (25 µg/ml) and chloramphenicol (25 µg/ml). Filter-sterilised SDS solution was added to the media to final concentration of 1 to 2% as indicated. Phenotypic growth tests in the presence of SDS were performed as follows: overnight cultures were diluted to OD 600 0.1 and 5 µl aliquots were spotted in a serial dilution series from 10 4 cells to 10 1 cells per 5 µl for Sco2149 TMD -AmiA and 5.10 6 to 10 5 for Sco3746 TMD -AmiA and PFD TMD -AmiA on LB agar supplemented with 1 or 2% SDS. Phenotypic testing for maltose fermentation employed the approach of (22)  Photographs of 96-well plates were captured as JPG files using a digital camera (DX AF-S NIKKOR 18-55 mm; Nikon) and colonies on agar with a digital scanner (EPSON perfection 3490 PHOTO). JPG files were imported into Gimp for cropping but otherwise were not processed.

Subcellular fractionation.
Membrane and cellular fractions were prepared as described by Keller et al. (22) with modifications. E. coli cells were grown overnight at 37°C in LB medium with appropriate antibiotics, subcultured and harvested at OD 600 of 0.2 for cells producing Sco2149 derivatives or OD 600 of 0.5 for cells producing Sco3746 and PFD derivatives. Cells producing Sco2149 constructs were resuspended in the same volume of hypertonic buffer (20mM Tris-HCl pH7.5/200mM NaCl) supplemented with EDTA-free protease inhibitor (Roche). Cells producing Sco3746 or PFD constructs were diluted to give a final OD 600 of 0.2 in the same buffer. Cells were then lysed by sonication (Branson Digital Sonifier 250) and the suspension was centrifuged for 10 min at 20 000 g at 4°C to remove unbroken cells and large cellular debris. The resulting supernatant was then ultracentrifuged for 1 hour at 220 000 g at 4°C to separate membrane and soluble fractions. An aliquot of the soluble fraction was kept for analysis and the membrane pellet was resuspended in 50mM Tris-HCl pH 7.5; 5mM MgCl 2 ; 10% (v/v) Glycerol. Protein concentration was estimated by the Lowry method (63) using the DCTM Protein Assay kit (Bio-Rad) and a standard curve generated with Bovine Serum Albumin (BSA). Membrane and soluble fractions were snap-frozen and kept at -20°C until further analysis. Urea extraction was undertaken as described previously (22).